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Original research
Activation of cryptic donor splice sites by non-coding and coding PAX6 variants contributes to congenital aniridia
  1. Maria Tarilonte1,2,
  2. Patricia Ramos1,
  3. Jennifer Moya1,
  4. Guilermo Fernandez-Sanz3,4,
  5. Fiona Blanco-Kelly1,2,
  6. Saoud Tahsin Swafiri1,2,
  7. Cristina Villaverde1,2,
  8. Raquel Romero1,
  9. Alejandra Tamayo1,2,
  10. Blanca Gener2,5,
  11. Patrick Calvas6,7,
  12. Carmen Ayuso1,2,
  13. Marta Corton1,2
  1. 1 Department of Genetics & Genomics, Instituto de Investigación Sanitaria - Fundación Jiménez Díaz University Hospital - Universidad Autónoma de Madrid (IIS-FJD-UAM), Madrid, Spain
  2. 2 Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain
  3. 3 Department of Ophthalmology, Fundación Jiménez Díaz University Hospital, Madrid, Spain
  4. 4 Department of Ophthalmology, Clínica Universidad de Navarra, Madrid, Spain
  5. 5 Department of Genetics, Cruces University Hospital, BioCruces Health Research Institute, Barakaldo, Spain
  6. 6 Service de Génétique Médicale, Hôpital Purpan, CHU Toulouse, Toulouse, France
  7. 7 INSERM U1056, Université Toulouse III, Toulouse, France
  1. Correspondence to Dr Marta Corton, Department of Genetics & Genomics, Instituto de Investigación Sanitaria - Fundación Jiménez Díaz University Hospital, Madrid 28040, Spain; mcorton{at}


Background The paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-function PAX6 variants.

Methods By locus-specific analysis of PAX6 using Sanger and/or targeted next-generation sequencing, we screened a Spanish cohort of 106 patients with PAX6-related diseases. Functional splicing assays were performed by in vitro minigene approaches or directly in RNA from patient-derived lymphocytes cell line, when available.

Results Five out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation.

Conclusion We describe new spliceogenic mechanisms for PAX6 variants mediated by creating or strengthening five different cryptic donor sites at exon 6. Our work revealed that the activation of cryptic PAX6 splicing sites seems to be a recurrent and underestimated cause of aniridia. Our findings pointed out the importance of functional assessment of apparently silent PAX6 variants to uncover hidden genetic alterations and to improve variant interpretation for genetic counselling in aniridia.

  • genetic screening/counselling
  • molecular genetics
  • ophthalmology
  • vision research
  • developmental

Data availability statement

All data relevant to the study are included in the article or uploaded as supplemental information.

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Data availability statement

All data relevant to the study are included in the article or uploaded as supplemental information.

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  • Contributors All the authors have substantially contributed to design, data acquisition, analysis or interpretation as well as to draft or revise the work and have approved the manuscript.

  • Funding This work was supported by several research grants from Spanish Institute of Health Carlos III (ISCIII) and the European Regional Development Fund (ERDF): PI17/01164, awarded to MC and the Spanish Centre for Biomedical Network Research on Rare Diseases (CIBERER, 06/07/0036 grant), awarded to CA; from the Ministry of Economy and Competitiveness/ERDF (MINECO, SAF2013-46943-R), the Spanish Mutua Madrileña Foundation and Spanish Foundation of Rare Diseases (FEDER), awarded to MC, as well as the Regional Government of Madrid (CAM, B2017/BMD3721), the University Chair UAM-IIS-FJD of Genomic Medicine and the Ramon Areces Foundation, awarded to CA. MC was sponsored by the ISCIII Miguel Servet Programme (CPII17_00006) and MT received a PhD fellowship from Conchita Rábago Foundation. JM and AT were sponsored by Regional Government of Madrid (CAM, PEJ-2017-AI/BMD-7256 and PEJD-2018-PRE/BMD-9453).

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.